Using an assay of T cell differentiation described in our laboratory (Rosenzweig, Blood 87, 1996 4040-4048), we have evaluated the ability of pig thymus to support primate T cell differentiation. Multiple experiments have confirmed our preliminary observation that fetal pig thymic stromal cultures can support T cell differentiation of rhesus CD34+ progenitor cells. These cultures yield CD3+ single positive cells (both CD4+ and CD8+) as well as double positive cells, with typically 50-75% of the cells being double positive cells. Detailed immunophenotyping of the cells resulting from these cultures has also revealed the presence of CD45RA+ cells predominantly in the single positive CD4 and double positive cells. In addition, we have also identified CD3+CD8+ TCR k cells. At least phenotypically, T cells arising in these cultures are similar to those obtained from rhesus thymic cultures and rhesus thymic tissue in vivo. Analysis of the proliferative capacity of T cells obtained from these cultures has confirmed that these cells respond to both CD3 cross-linking and lectin stimulation. In addition we have also established that, as expected, T cells derived from swine thymic stromal cultures are susceptible to SIV infection. The number of T cells obtained from pig thymic stromal cultures can be increased by the hematopoietic growth factor flt3-ligand, suggesting that this cytokine may prove useful in enhancing T cell differentiation in transplanted pig thymus in vivo. Initial studies with human CD34 bone marrow cells have confirmed that swine thymic tissue can also support T cell differentiation of human progenitor cells. Future studies will examine the ability of rhesus cells to increase the efficiency of T cell differentiation supported by pig thymic tissue. These studies should provide information to support studies of transplantation of pig thymic tissue in SIV-infected macaques and may lead to novel approaches to reconstitute immune function in HIV-infected people.